This invention relates to a novel transcriptional regulatory factor comprising bromodomains and the encoding gene.
The bromodomain is a characteristic amino-acid motif seen in transcriptional regulatory factors and is believed to be involved in the interactions with other transcriptional regulatory factors. Proteins comprising the bromodomain, normally have one or two (Tamkun et al. (1992) Nuc. Acids Res. 20:2603; Haynes et al. (1992) Nuc. Acids Res. 20: 2603), but as many as five (Nicolas et al. (1996) Gene 175(12):233-240) bromodomain motifs. This motif has been identified in a wide range of animals, for example, in the homeotic gene (Digan et al. (1986) Dev. Biol. 114:161-169; Tamkun et al. (1992) Cell 68: 561-572) of the fruit fly (Drosophila), in the transcriptional regulatory genes of yeasts (Winston et al. (1987) Genetics 115:649-656; Laurent et al. (1991) Proc. Nat. Acad. Sci. USA 88:2687-2691) and in mammals (Denis et al. (1996) Genes and Devel. 10:261-271; Yang et al. (1996) Nature 382:319-324). According to a recent report (Jeanmougin et al. (1997) Trends Biochem. Sci. 22:151-153), 37 bromodomain genes, including 13 human genes are recorded in the database. In addition to the bromodomain motif of amino acid residues 59-63, the sequences adjacent to the motif are also structurally conserved, and furthermore, 4 xcex1-helixes (Z, A, B, and C) are reported to be coded within the long 110 amino acids.
When these bromodomain-comprising transcriptional regulatory factors are compared, they all regulate signal-dependent transcription in actively proliferating cells (Tamkun et al. (1992) Cell 68:561-572; Haynes et al. (1992) Nuc. Acids Res. 20:2603). This characteristic implies that oncogenesis may occur when a gene encoding a bromodomain-containing protein undergoes abnormal regulation. In reality, six bromodomain genes have been experimentally proven to associate with oncogenesis. Three of these genes HRX/ALL-1 (Tkachuk et al. (1992) Cell 71:691-700 ; Gu et al. (1992) Cell 71:701-708); TIF1 (Miki et al. (1991) Proc. Nat. Acad. Sci. USA 88:5167-5171; Le Douarin et al. (1995) EMBO J. 14:2020-2033) and CBP (Borrow et al. (1996) Nature Genet. 14:33-41) are linked with the gene cleavage points in leukemia. All three of these proteins contain the C4HC3 (also called PHD/LAP/TRX) zinc-finger (Aasland et al. (1995) Trends Biochem. Sci. 20:56-59; Koken et al. (1995) CR Acad. Sci. III, 318:733-739; Saha et al. (1995) Proc. Nat. Acad. Sci. USA 92:9737-9741). Also, there are findings that CBP/P300 interact with p53 (Gu et al. (1997) Nature 387:819-823; Lill et al.(1997) Nature 387:823-827) and other various transcriptional factors, suggesting that CBP and the homologous gene P300 play a key-role in cancer.
The other three genes have been suggested to be linked with cancer in various ways. BRG1 interacts with retinoblastoma protein RB (Dunaief et al. (1994) Cell 79:119-130), inducing formation of flat, growth-arrested cells, and thereby showing a tumor-suppressive activity. RING3 has a homology with the fruit fly (Drosophila) growth control protein fsh (Haynes et al. (1989) Dev. Biol. 134:246-257) and is a serine-threonine kinase having endonuclear autophosphorylation activity. This activity has been reported to be linked to the growth phase of chronic and acute lymphocytic leukemia (Denis et al. (1996) Genes and Devel. 10:261-271). As for P/CAF, it has been reported to inhibit the interaction between E1A and p300/CBP (Yang et al. (1996) Nature 382:319-324). When P/CAF is exogenously expressed on HeLa cells, the cell cycle is inhibited. This is believed to be due to the disruption of the transcriptional regulation of E1A by the binding of P/CAF to p300/CBP. Similar to p300/CBP (Bannister and Kouzarides (1996) Nature 384:641-643), P/CAF has been reported to contain histone acetyl-transferase activity (Yang et al. (1996) Nature 382:319-324).
Thus, regulatory abnormalities of transcriptional regulatory factors comprising bromodomains are envisaged to be closely associated with various diseases, particularly, cancer and other cell-proliferation-linked diseases. Hence, attention has been focused on transcriptional regulatory factors comprising bromodomains in the recent years as novel targets for the treatment of cancer and other cell-proliferation-linked diseases.
The present invention provides a novel transcriptional regulatory factor comprising bromodomains, the encoding gene, a method of production, and a screening method for a drug-candidate compound that utilizes the protein and the gene of the present invention.
In order to solve the above-mentioned problems, EST databases were BLAST searched using various nucleotide sequences encoding known bromodomain motifs. As a result, several potential bromodomain-gene-encoding ESTs were found by the search using nucleotide sequence of Tetrahymena thermophila HAT A1 gene. One of these ESTs, the fetal lung cDNA library-derived EST (W17142) was found to encode an unknown gene. Therefore, isolation of full-length cDNA of EST W17142 was initiated. Specifically, primers were designed based on the EST W17142 sequence, and an amplification product was obtained by the polymerase chain reaction using testicular cDNA as the template. Then, the testicular cDNA library was screened using this amplification product as the probe, and a re-screening of the library was done using the cDNA clone comprising the above-mentioned EST sequence, thereby successfully isolating a full-length cDNA corresponding to EST W17142. By structural analysis of the protein encoded by the isolated cDNA, the present Inventors found that apart from the bromodomain, said protein had several regions and domains conserved in transcriptional regulatory factors.
Also, they found that the protein encoded by the isolated cDNA interacts with hSNF2H and hSNF2L that are implicated in the series of processes related to the chromatin-mediated transcriptional regulatory mechanism, and also with the transcription co-activator NcoA-62/Skip, which interacts with the ligand-binding domains of various nuclear receptors (VDR, RAR) and the Ski viral oncoprotein.
The transcriptional regulatory factor and the encoding gene revealed by the Inventors can be utilized for the screening of compounds inhibiting the binding between said transcriptional regulatory factor and an interacting factor, and compounds which regulate the binding activity. The compounds thus isolated are expected to be applied as pharmaceuticals.
Namely, the present invention relates to a novel transcriptional regulatory factor comprising a bromodomain and the encoding gene, as well as methods of production, and a screening method for related-factors and drug-candidate compounds that utilize the protein and the gene of the present invention. Specifically, the present invention relates to:
1. a protein comprising the amino acid sequence of SEQ ID NO:1 or 10;
2. a transcriptional regulatory factor comprising a bromodomain and the amino acid sequence of SEQ ID NO:1 or 10, wherein one or more amino acids are replaced, deleted, added, and/or inserted;
3. a protein comprising the amino acid sequence of SEQ ID NO:1 or 10 wherein one or more amino acids are replaced, deleted, added, and/or inserted, and having an activity to bind to a protein selected from the group consisting of hSNF2H,hSNF2L,NCoA-62/Skip and homologues thereof;
4. a transcriptional regulatory factor comprising a bromodomain, and encoded by a DNA hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO:2 or 9;
5. a transcriptional regulatory factor encoded by a DNA hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO:2 or 9, and having an activity to bind to a protein selected from the group consisting of hSNF2H, hSNF2L,NCoA-62/Skip and homologues thereof;
6. a DNA encoding the transcriptional regulatory factor of any one of (1) to (5);
7. the DNA of (6), which contains the coding region of the nucleotide sequence of SEQ ID NO:2 or 9;
8. a vector containing the DNA of (6) or (7);
9. a transformant carrying, in an expressible manner, the DNA of (6) or (7);
10. a method for producing the transcriptional regulatory factor of any one of (1) to (5), the method comprising culturing the transformant of (9);
11. an antibody which binds to the transcriptional regulatory factor of any one of (1) to (5);
12. a method for screening a compound having an activity to bind to the transcriptional regulatory factor of any one of (1) to (5), the method comprising the steps of,
(a) exposing a test sample to said transcriptional regulatory factor,
(b) detecting the binding activity between the test sample and said transcriptional regulatory factor, and,
(c) selecting a compound having the binding activity to said transcriptional regulatory factor;
13. a method for screening a compound which promotes or inhibits the binding between the transcriptional regulatory factor of any one of (1) to (5) and a protein selected from the group consisting of hSNF2H, hSNF2L, NCoA-62/Skip and homologues thereof, the method comprising the steps of,
(a) exposing the transcriptional regulatory factor to hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof, in the presence of the test sample,
(b) detecting the binding activity between said transcriptional regulatory factor and hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof,
(c) selecting a compound which increases or decreases said binding activity when compared with the binding activity in the absence of the test sample (control);
14. a compound which is obtainable by the method of (13), which inhibits the binding between the transcriptional regulatory factor of any one of (1) to (5) and a protein selected from the group consisting of hSNF2H, hSNF2L, NCoA-62/Skip and homologues thereof; and
15. a DNA comprising at least 15 nucleotides, which can specifically hybridize with the DNA comprising the nucleotide sequence of SEQ ID NO:2 or 9. The DNA can also be at least 351, 400, 450, 500, 700, 1000, 2200, 2500, or 3000 bp in length.
Herein, xe2x80x9ctranscriptional regulatory factorxe2x80x9d indicates a protein that regulates gene expression. xe2x80x9cBromodomainxe2x80x9d means, an amino acid motif associated with protein-protein interactions conserved within transcriptional regulatory factors linked to signal-dependent transcription.
The present invention relates to a transcriptional regulatory factor comprising a bromodomain. The amino acid sequences of the protein named xe2x80x9cTCoA1xe2x80x9d included in the present invention, and its variant are shown in SEQ ID NO:1 and SEQ ID NO:10, respectively, and the nucleotide sequences of their cDNA in SEQ ID NO:2 and SEQ ID NO:9, respectively (unless otherwise noted, these will be grouped as xe2x80x9cTCoA1xe2x80x9d, hereafter). xe2x80x9cTCoA1xe2x80x9d is most deeply associated with the presumed proteins of nematode (C. elegans) chromosome III genes F26H11.2, F26H11.3a and F26H11.3b (Wilson et al. (1994) Nature 368:32-38), the function of which are unknown and which were identified by the genomic sequence of one cosmid F26H11. When the amino acid sequence of these two proteinsxe2x80x94the presumed nematode protein and the xe2x80x9cTCoA1xe2x80x9d proteinxe2x80x94are compared, although the domain configurations are different, they are extremely alike.
Like many bromodomain proteins, xe2x80x9cTCoA1xe2x80x9d has one bromodomain. Being structurally similar to the TIF family, GCN5 and P/CAF, this bromodomain is situated close to the carboxyl-terminus (Jeanmougin et al. (1997) Trends Biochem. Sci. 22:151-153). Like other bromodomain proteins, xe2x80x9cTCoA1xe2x80x9d has a C4HC3 zinc-finger. The combination of the bromodomain and the zinc-finger has been discovered frequently in the gene cleavage points in several leukemia, so far (Tkachuk et al. (1992) Cell 71:691-700; Gu et al. (1992) Cell 71: 701-708; Miki et al. (1991) Proc. Nat. Acad. Sci. USA 88:5167-5171; Le Douarin et al. (1995) EMBO J. 14:2020-2033; Borrow et al. (1996) Nature Genet. 14:33-41). Therefore, xe2x80x9cTCoA1xe2x80x9d is a candidate cleavage gene associated with chromosome no. 17 q23.
xe2x80x9cTCoA1xe2x80x9d has numerous nuclear transport signal motifs. This indicates that xe2x80x9cTCoA1xe2x80x9d protein is located within the nucleus. Like other bromodomain proteins, xe2x80x9cTCoA1xe2x80x9d has a LXXLL motif series that likely determines the site of interaction with nuclear receptors (Heery et al. (1997) Trends Biochem. Sci. 22:151-153; Torchia et al. (1997) Nature 387:677-684). The possibility that it interacts with the receptor bound to a ligand via the LXXLL domain indicates that xe2x80x9cTCoA1xe2x80x9d functions as a transcriptional co-activator. In the carboxyl terminus of xe2x80x9cTCoA1xe2x80x9d, a glutamine-rich domain is located spanning a very large region. Glutamine-rich domains have been identified in many transcriptional regulatory factors including bromodomain-containing proteins like p300/CBP (Shikama et al. (1997) Trends in Cell Biol. 2:230-236) and fsh protein of fruit fly (Drosophila) (Haynes et al. (1989) Dev. Biol. 134:246-257). These acidic regions have been predicted to be associated with the protein-protein interactions that determine the function as an active substance (Courey et al. (1989) Cell 59:827-836).
xe2x80x9cCoA1xe2x80x9d protein has many common characteristics with other bromodomain proteins known to be linked to cell-proliferation-linked diseases such as cancer. Therefore, xe2x80x9cTCoA1xe2x80x9d protein may also be linked to cancer, and thus, the xe2x80x9cTCoA1xe2x80x9d protein and its gene, a compound that regulate the function of the xe2x80x9cTCoA1xe2x80x9d protein can be applied for the prevention and treatment of cancer and other cell-proliferation-linked diseases.
Moreover, the fact that hSNF2H and hSNF2L, which interact with xe2x80x9cTCoA1xe2x80x9d, are involved in the series of processes related to the chromatin-mediated transcriptional regulatory mechanism, strongly indicates that xe2x80x9cTCoA1xe2x80x9d is playing some sort of a role in chromatin-mediated transcriptional regulation. Therefore, it can be conceived that xe2x80x9cTCoA1xe2x80x9d is playing a major role as a protein that integrates transcriptional responses towards nuclear receptors by associating with the chromatin reconstruction mechanism.
The transcriptional regulatory factor of the present invention can be prepared by methods known to one skilled in the art, as a recombinant protein made using genetic engineering techniques, and also as a natural protein. For example, a recombinant protein can be prepared by inserting DNA encoding the protein of the present invention (for example, DNA comprising the nucleotide sequence of SEQ ID NO:2 or 9) into a suitable expression vector, introducing this into a host cell, and purifying the protein from the resulting transformant. The natural protein can be acquired by preparing a column coupled with an antibody obtained by immunizing a small animal with the recombinant protein, and performing affinity chromatography for extracts of tissues or cells (for example, testis, tumor cells, etc.) expressing high levels of the transcriptional regulatory factor of the present invention.
Also, this invention features a transcriptional regulatory factor, which is functionally equivalent to the xe2x80x9cTCoA1xe2x80x9d protein (SEQ ID NO:1 or 10). This transcriptional regulatory factor includes, mutants of the xe2x80x9cTCoA1xe2x80x9d protein (SEQ ID NO:1 or 10) and xe2x80x9cTCoA1xe2x80x9d proteins obtained from various living organisms.
To isolate a protein functionally equivalent to a certain protein, the method of inserting a mutation into the amino acids within the protein is well known to one skilled in the art. In other words, for a person skilled in the art, the isolation of a transcriptional regulatory factor functionally equivalent to the xe2x80x9cTCoA1xe2x80x9d protein, is a standard procedure which can be done using, for example, the PCR-mediated, site-directed-mutation-induction system (GIBCO-BRL, Gaithersburg, Md.), oligonucleotide-mediated, sight-directed-mutagenesis (Kramer et al. (1987) Methods in Enzymol. 154:350-367) suitably replacing amino acids that do not influence the function of the xe2x80x9cTCoA1xe2x80x9d protein set forth in SEQ ID NO:1 or 10. Mutations of amino acids can occur spontaneously as well. The transcriptional regulatory factor of the invention includes those comprising the amino acid sequence of xe2x80x9cTCoA1xe2x80x9d protein in SEQ ID NO:1 or 10 in which one or more amino acids have been replaced, deleted, added, and/or inserted, and have a binding-activity with hSNF2H, hSNF2L and NcoA-62/Skip, and those comprising the amino acid sequence of xe2x80x9cTCoA1xe2x80x9d protein in SEQ ID NO:1 or 10 in which one or more amino acids have been replaced, deleted, added, and/or inserted, and comprise a bromodomain.
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. The substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
The number of amino acids that are mutated is not particularly restricted, as long as the function of the xe2x80x9cTCoA1xe2x80x9d protein is maintained. Normally, it is within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids and even more preferably within 3 amino acids. The site of mutation may be any site, as long as the function of the xe2x80x9cTCoA1xe2x80x9d protein is maintained.
Proteins having amino acid sequences modified by deleting, adding and/or replacing one or more amino acid residues of a certain amino acid sequence, have been known to retain the original biological activity (Mark et al., Proc. Natl. Acad. Sci. USA (1984) 81:5662-5666; Zoller et al. Nucleic Acids Research (1982) 10:6487-6500; Wang et al., Science 224:1431-1433; Dalbadie-McFarland et al., Proc. Natl. Acad. Sci. USA (1982) 79:6409-6413).
As for the amino acid residue to be mutated, it is preferable to be mutated into a different amino acid in which the properties of the amino acid side-chain are conserved. Examples of properties of amino acid side chains are, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and amino acids comprising the following side chains: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W). (The parenthetic letters indicate the one-letter codes of amino acids). A xe2x80x9cconservative amino acid substitution is a replacement of one amino acid belonging to one of the above groups with another amino acid in the same group.
In the present invention, the protein having several deletions in the amino acid sequence of the xe2x80x9cTCoA1xe2x80x9d protein (SEQ ID NO:1 or 10) includes a partial peptide comprising binding-activity with hSNF2H, hSNF2L, NcoA-62/Skip or homologues thereof. As described in Example 6 (FIG. 5), the N-terminus of the xe2x80x9cTCOA1xe2x80x9d protein has a binding-activity with hSNF2H, hSNF2L, NcoA-62/Skip or homblogues thereof. Peptides such as these, inhibit the binding between xe2x80x9cTCoA1xe2x80x9d protein and the above binding-proteins in vivo, and thus can be used to inhibit the functions of the xe2x80x9cTCoA1xe2x80x9d protein in vivo.
A fusion protein including the xe2x80x9cTCoA1xe2x80x9d protein can be given as an example of a protein into which several amino acid residues have been added to the amino acid sequence of the xe2x80x9cTCoA1xe2x80x9d protein (SEQ ID NO:1 or 10). Fusion proteins are, fusions of the xe2x80x9cTCoA1xe2x80x9d protein and other peptides or proteins, and are included in the present invention. Fusion proteins can be made by techniques well known to a person skilled in the art, such as by linking the DNA encoding the xe2x80x9cTCoA1xe2x80x9d protein of the invention with DNA encoding other peptides or proteins, so as the frames match, inserting this into an expression vector and expressing it in a host. There is no restriction as to the peptides or proteins fused to the protein of the present invention.
Known peptides, for example, FLAG (Hopp et al., Biotechnology (1988) 6:1204-1210), 6xc3x97His containing six His (histidine) residues, 10xc3x97His, Influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, xcex1-tubulin fragment, B-tag, Protein C fragment, and such, can be used as peptides that are fused to the protein of the present invention. Examples of proteins that are fused to protein of the invention are, GST (glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulin constant region, xcex2-galactosidase, MBP (maltose-binding protein), and such.
Fusion proteins can be prepared by fusing commercially available DNA encoding these peptides or proteins with the DNA encoding the protein of the present invention and expressing the fused DNA prepared.
The hybridization technique (Sambrook et al., Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is well known to one skilled in the art as an alternative method for isolating a protein functionally equivalent to a certain protein. In other words, for a person skilled in the art, it is a general procedure to obtain a transcriptional regulatory factor functionally equivalent to the xe2x80x9cTCoA1xe2x80x9d protein, by isolating DNA having a high homology with the whole or part of the DNA encoding the xe2x80x9cTCoA1xe2x80x9d protein of SEQ ID NO:2 using the hybridization technique. The transcriptional regulatory factor of the present invention, includes transcriptional regulatory factors comprising bromodomains which are encoded by the DNA hybridizing with the DNA encoding xe2x80x9cTCoA1xe2x80x9d protein of SEQ ID NO:2. Animals which can be used to isolate a functionally equivalent transcriptional regulatory factor are, apart from humans, for example, mice, rats, cattle, monkeys and pigs, but there are no restrictions to the animal used. The stringency of hybridization is defined as equilibrium hybridization under the following conditions: 42xc2x0 C., 2xc3x97SSC, 0.1% SDS (low stringency); 50xc2x0 C., 2xc3x97SSC, 0.1% SDS (medium stringency); and 65xc2x0 C., 2xc3x97SSC, 0.1% SDS (high stringency). If washings are necessary to achieve equilibrium, the washings are performed with the hybridization solution for the particular stringency desired. In general, the higher the temperature, the higher is the homology between two strands hybridizing at equilibrium. However, several factors other than temperature can influence the stringency of hybridization and one skilled in the art can suitably select the factors to accomplish a similar stringency.
In place of hybridization, the gene amplification method using a primer synthesized based on the sequence information of the DNA sequence of SEQ ID NO:9 encoding the xe2x80x9cTCoA1xe2x80x9d protein, for example, the polymerase chain reaction (PCR) method can be utilized to isolate a DNA encoding a transcriptional regulatory factor functionally equivalent to the xe2x80x9cTCoA1xe2x80x9d protein.
Proteins encoded by the DNA isolated through the above hybridization technique or gene amplification techniques, normally have a high homology to the amino acid sequence of the xe2x80x9cTCoA1xe2x80x9d protein. xe2x80x9cHigh homologyxe2x80x9d refers to, normally a homology of 40% or higher, preferably 60% or higher, more preferably 80% or higher, even more preferably 95% or higher with the amino acid sequence of the xe2x80x9cTCoA1xe2x80x9d protein. The homology of a protein can be determined by following the algorithm in xe2x80x9cWilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80, 726-730xe2x80x9d.
The xe2x80x9cpercent identityxe2x80x9d of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3. Where gaps exist between two sequences, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
Transcriptional regulatory factors functionally equivalent to the xe2x80x9cTCoA1xe2x80x9d protein (SEQ ID NO:1 or 10) isolated by the above hybridization technique or gene amplification techniques include, those having a binding activity with hSNF2H, hSNF2L and NcoA-62/Skip, and a high homology in the primary structure with the xe2x80x9cTCoA1xe2x80x9d protein (SEQ ID NO:1 or 10), and those having the bromodomain, which is a motif thought to be vital to the function linked with cancer, and a high homology in the primary structure with the xe2x80x9cTCoA1xe2x80x9d protein (SEQ ID NO:10).
Other than the bromodomain, these transcriptional regulatory factors also comprise sequences involved in the interactions with other proteins (for example, leucine-zipper, LXXLL motif), sequences involved in the binding with DNA (for example, zinc-finger), and nuclear transport signals.
The existence of the bromodomain within a protein can be determined by searching the bromodomain motif PROSITE database on DNASIS (Hitachi Software Engineering).
This invention also relates to a DNA encoding the above transcriptional regulatory factor. There is no restriction as to the DNA of the present invention as long as it encodes the transcriptional regulatory factor of the invention, and includes cDNA, genomic DNA and chemically synthesized DNA. Also as long as they can encode the protein of the invention, DNAs comprising arbitrary sequences based on the degeneracy of the genetic code are also included. cDNA encoding the protein of the invention can be prepared, for example, by preparing a primer based on nucleotide information (for example, SEQ ID NO:9) of DNA encoding the transcriptional regulatory factor of the invention and performing plaque PCR (for example please refer, Affara NA et al. (1994) Genomics 22:205-210). In the case of genomic DNA, preparation can be done for example, by the method using commercially available xe2x80x9cQiagen genomic DNA kitsxe2x80x9d (Qiagen, Hilden, Germany). The nucleotide sequence of the DNA acquired can be decided by ordinary methods in the art by using, for example, the commercially available xe2x80x9cdye terminator sequencing kitxe2x80x9d (Applied Biosystems). The DNA of the present invention, as stated later, can be utilized for the production of a recombinant protein and gene therapy.
The present invention also features a vector into which the DNA of the present invention has been inserted. There is no restriction as to the vector to which DNA is inserted, and various vectors such as those for expressing the transcriptional regulatory factor of the present invention in vivo and those for preparing the recombinant protein can be used according to the objective. To express the transcriptional regulatory factor of the present invention in vivo (especially for gene therapy), various viral vectors and non-viral vectors can be used. Examples of viral vectors are, adenovirus vectors (pAdexLcw) and retrovirus vectors (pZlPneo), etc. Cationic liposomes can be given as examples of non-viral vectors. Expression vectors are especially useful when using for the purpose of producing the transcriptional regulatory factor of the invention. For example, when using colibacili (E. coli) the xe2x80x9cpREP4xe2x80x9d (Qiagen, Hilden, Germany) and such vectors, when using yeast xe2x80x9cSP-Q01xe2x80x9d (Stratagene, La Jolla, Calif.) and such, when using insect cells xe2x80x9cBac-to-Bac 5 baculovirus expression systemxe2x80x9d (GIBCO-BRL, Gaithersburg, Md.) are highly appropriate, but there is no restriction. Also, when using mammalian cells such as CHO cells, COS cells, NIH3T3 cells, for example, the xe2x80x9cLacSwitch II expression system (Stratagene, La Jolla, Calif.) is highly suitable, but there is no restriction. Insertion of the DNA of the present invention into a vector can be done using ordinary methods in the art.
The present invention also refers to a transformant, carrying, in an expressible manner, the DNA of the present invention. The transformant of the present invention includes, those carrying the above-mentioned vector into which DNA of the present invention has been inserted, and those having host genomes into which the DNA of the present invention has been integrated. As long as the DNA of the present invention is maintained in an expressible manner, no distinction is made as to the form of existence of the transformants. There is no particular restriction as to the cells into which the vector is inserted. For example, when using for the purpose of gene therapy, various cells can be used as target cells according to the type of disease. Also, when the purpose is to produce the transcriptional regulatory factor of the present invention, for example, E. coli, yeast, animal cells and insect cells can be used as hosts. Introduction of a vector into a cell can be done using known methods such as electroporation and calcium phosphate method.
Common methods applied in the art may be used to isolate and purify said recombinant protein from the transformant made for the production of recombinant proteins.
For example, after collecting the transformant and obtaining the extracts, the objective protein can be purified and prepared by, ion exchange chromatography, reverse phase chromatography, gel filtration, or affinity chromatography where an antibody against the protein of the present invention has been immobilized in the column, or by combining several of these columns.
Also when the protein of the present invention is expressed within host cells (for example, animal cells and E. coli) as a fusion protein with glutathione-S-transferase protein or as a recombinant protein supplemented with multiple histidines, the expressed recombinant protein can be purified using a glutathione column or nickel column. After purifying the fusion protein, it is also possible to exclude regions other than the objective protein by cutting with thrombin or factor-Xa as required.
The present invention also features an antibody binding to the transcriptional regulatory factor of the invention. There is no particular restriction as to the form of the antibody of the present invention and include, apart from polyclonal antibodies, monoclonal antibodies as well. The antiserum obtained by immunizing animals such as rabbits with the transcriptional regulatory factor of the present invention, polyclonal and monoclonal antibodies of all classes, humanized antibodies made by genetic engineering, human antibodies, are also included. The antibodies of the present invention can be prepared by the following methods. Polyclonal antibodies can be made by, obtaining the serum of small animals such as rabbits immunized with the transcriptional regulatory factor of the present invention, attaining a fraction recognizing only the transcriptional regulatory factor of the invention by an affinity column coupled with the protein of the present invention, and purifying immunoglobulin G or M from this fraction by a protein G or protein A column. Monoclonal antibodies can be made by immunizing small animals such as mice with the transcriptional regulatory factor of the present invention, excising the spleen from the animal, homogenizing the organ into cells, fusing the cells with mouse myeloma cells using a reagent such as polyethylene glycol, selecting clones that produce antibodies against the transcriptional regulatory factor of the invention from the fused cells (hybridomas), transplanting the obtained hybridomas into the abdominal cavity of a mouse, and extracting ascites. The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which the transcriptional regulatory factor of the present invention is coupled. The antibody of the invention can be used for purifying and detecting the transcriptional regulatory factor of the invention. It can also be used as a pharmaceutical drug to inhibit the function of the present transcriptional regulatory factor. When using the antibody as a drug, in the view-point of immunogenicity, human antibodies or humanized antibodies are effective. The human antibodies or humanized antibodies can be prepared by methods commonly known to one skilled in the art. For example, human antibodies can be made by, immunizing a mouse whose immune system has been changed to that of humans, with the transcriptional regulatory factor of the invention. Also, humanized antibodies can be prepared by, for example, cloning the antibody gene from monoclonal antibody producing cells and using the CDR graft method which transplants the antigen-recognition site of the gene into a known human antibody.
The present invention also relates to a method for screening a compound that binds to the transcriptional regulatory factor of the invention. The screening method of the invention includes the steps of, (a) exposing a test sample to the transcriptional regulatory factor of the invention, (b) detecting the binding activity between the test sample and the transcriptional regulatory factor of the invention, and (c) selecting a compound having an activity to bind to the transcriptional regulatory factor of the invention. Any test sample can be used for the screening without particular restrictions. Examples are, cell extracts, culture supernatants, synthetic low molecular weight compound libraries, purified proteins, expression products of gene libraries, synthetic peptide libraries, and so on.
Isolation of a compound that binds to the transcriptional regulatory factor using said transcriptional regulatory factor can be done using methods commonly known to one skilled in the art. The screening of a protein which binds to the transcriptional regulatory factor of the invention can be done by, for example, creating a cDNA library from tissues or cells (for example, testis tissue cells and tumor cell lines) expected to express a protein binding to the transcriptional regulatory factor of the invention using a phage vector (xcexgt11 and Zap, etc.), expressing this cDNA library on LB-agarose, fixing the expressed proteins on the filter, biotin-labeling the transcriptional regulatory factor of the invention or purifying it as a fusion protein with GST protein, reacting this with the above-described filter, and detecting plaques expressing the binding proteins using streptavidin or anti-GST antibody (West Western Blotting method) (Skolnik et al. (1991) Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases, Cell 65:83-90). The screening of a protein binding to the transcriptional regulatory factor of the invention or its gene, can also be done by following xe2x80x9cthe two-hybrid systemxe2x80x9d (xe2x80x9cMATCHMAKER Two-hybrid Systemxe2x80x9d, xe2x80x9cMammalian MATCHMAKER Two-Hybrid Assay Kitxe2x80x9d, xe2x80x9cMATCHMAKER One-Hybrid Systemxe2x80x9d (Clontech), xe2x80x9cHybriZAP Two-Hybrid Vector Systemxe2x80x9d (Stratagene), or Referencexe2x80x94xe2x80x9cDalton S, and Treisman R (1992) Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element. Cell 68, 597-612xe2x80x9d). In the two-hybrid system, the transcriptional regulatory factor of the invention is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells. A cDNA library, is prepared from cells expected to express a protein binding to the transcriptional regulatory factor of the invention, in a way that the library is expressed in the form of being fused to the VP16 or GAL4 transcriptional activation region. The cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the transcriptional regulatory factor of the invention is expressed in yeast cells, the binding of the two activates a reporter gene making positive clones detectable). A protein binding to the transcriptional regulatory factor of the invention can be recovered by, introducing the cDNA isolated above to E. coli and expressing the protein encoded by said cDNA.
Also, a protein binding to the transcriptional regulatory factor of the invention can be screened by, applying the culture supernatants or cell extracts of cells expected to express a protein binding to the transcriptional regulatory factor of the invention onto an affinity column in which the protein of the invention is immobilized and purifying the protein that binds specifically to the column.
The method of screening molecules that bind when the immobilized transcriptional regulatory factor of the invention is exposed to synthetic chemical compounds, or natural substance banks, or a random phage peptide display library, or the method of screening using high-throughput based on combinatorial chemistry techniques (Wrighton et al., Small peptides as potent mimetics of the protein hormone erythropoietin, Science (UNITED STATES) (1996), 273:458-464; Verdine G. L., The combinatorial chemistry of nature, Nature (ENGLAND) (1996) 384:11-13; Hogan J. C., Jr., Directed combinatorial chemistry. Nature (ENGLAND) (1996) 384:17-19) to isolate low molecular weight compounds, proteins (or their genes) and peptides are methods well known to one skilled in the art.
A biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound compound in the present invention. When such a biosensor is used, the interaction between the protein of the invention and a test compound can be observed real-time as a surface plasmon resonance signal, using only a minute amount of proteins without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the transcriptional regulatory factor of the invention and a test compound using a biosensor such as BIAcore.
The present invention also relates to a method for screening a compound able to promote or inhibit the binding between the transcriptional regulatory factor of the invention and an interacting-protein. Detection of a binding between the TCoA1 protein and hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof enabled such a screening. This screening can be done using the method comprising the steps of: (a) exposing the transcriptional regulatory factor of the invention to hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof, under the presence of a test sample; (b) detecting the binding activity between the transcriptional regulatory factor of the invention and hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof; and (c) selecting a compound which decreases said binding-activity when compared with the assay in the absence of a test sample (control).
There are no particular restrictions as to the test sample used. Examples are, cell extracts, culture supernatants, libraries of synthetic low molecular weight compounds, purified proteins, expression products of gene libraries, synthetic peptide libraries, etc. The compound isolated by the above-described screening of a protein binding to the protein of the invention may also be used as a test sample.
The transcriptional regulatory factor of the invention used for the screening may be a whole protein or a partial peptide comprising binding regions with hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof. hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof used for the screening may be whole proteins or partial peptides comprising binding regions with the transcriptional regulatory factor of the invention.
The detection of the binding activity between the transcriptional regulatory factor of the invention and hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof, can be performed, for example, as follows.
A test sample and hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof is added to the transcriptional regulatory factor of the invention immobilized on a microplate, reacted with a mouse or rabbit antibody against hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof, further reacted with an anti-mouse or anti-rabbit antibody labeled with peroxidase, alkaline phosphatase and such, a labeled enzyme substrate is added and the enzyme activity is measured. Compounds that show an enzyme activity that is lower to or higher than that in the absence of a test sample, are selected. Thereby, compounds having an activity to promote or inhibit the binding between the transcriptional regulatory factor of the invention and hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof are obtained.
This screening may be performed also by, using hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof as the immobilized protein, and the transcriptional regulatory factor of the invention as the protein that is added with the test sample.
Also, the transcriptional regulatory factor of the invention or hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof added together with the test sample may be directly labeled with peroxidase, or alkaline phosphatase, or used as a fusion protein with such enzymes. Compounds having an activity that activates or inhibits the binding between the transcriptional regulatory factor of the invention and hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof may also be selected by, expressing as fusion proteins with enzymes other than the above, such as, luciferase, xcex2-galactosidase, or GFP protein and measuring the inhibition or promotion of the enzyme activity by a test sample.
The mammalian two-hybrid system (Clontech, Palo Alto) can also be used to screen a compound that promotes or inhibits the binding between the transcriptional regulatory factor of the invention and an interacting-protein. Namely, using the two-hybrid system, the transcriptional regulatory factor of the invention and an interacting-protein is expressed in mammalian cells, a test sample is added to said mammalian cells, and then reporter-activity is measured. The detected reporter-activity is compared, and compounds that give a value that is lower to or higher than the reporter-activity in the absence of a test sample, are selected. Thus, a compound that promotes or inhibits the binding between the transcriptional regulatory factor of the invention and hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof can be obtained.
A compound screened by the screening of the invention may be applied for the prevention and treatment of cancer and other cell-proliferation-linked diseases. When using the isolated compound as a pharmaceutical for humans and other mammals, such as, mice, rats, guinea-pigs, rabbits, chicken, cats, dogs, sheep, pigs, monkeys, baboons, chimpanzees, the isolated compound can be directly administered or can be formulated into a dosage form using known pharmaceutical preparation methods. For example, according to the need, the drugs can be taken orally as sugar-coated tablets, capsules, elixirs and microcapsules or non-orally in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid. For example, the compounds can be mixed with pharmacologically acceptable carriers or medium, specifically, sterilized water, physiological saline, plant-oil, emulsifiers, solvents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives and binders, in a unit dose form required for generally accepted drug implementation. The amount of active ingredients in these preparations makes a suitable dosage within the indicated range acquirable.
Examples for additives which can be mixed to tablets and capsules are, binders such as gelatin, corn starch, tragacanth gum and arabic gum; excipients such as crystalline cellulose; swelling agents such as corn starch, gelatin and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose or saccharin; flavoring agents such as peppermint, Gaultheria adenothrix oil and cherry. When the unit dosage form is a capsule, a liquid carrier, such as oil, can also be included in the above ingredients. Sterile composites for injections can be formulated following normal drug implementations using vehicles such as distilled water used for injections.
Physiological saline, glucose, and other isotonic liquids including adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used as aqueous solutions for injections. These can be used in conjunction with suitable solubilizers, such as alcohol, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, non-ionic surfactants, such as Polysorbate 80 (TM) and HCO-50.
Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may be used in conjunction with benzyl benzoate or benzyl alcohol as a solubilizers; may be formulated with a buffer such as phosphate buffer and sodium acetate buffer; a pain-killer such as procaine hydrochloride; a stabilizer such as benzyl alcohol, phenol; and an anti-oxidant. The prepared injection is filled into a suitable ampule.
Methods well known to one skilled in the art may be used to administer a pharmaceutical compound to patients, for example as intraarterial, intravenous, percutaneous injections and also as intranasal, transbronchial, intramuscular or oral administrations. The dosage and method of administration vary according to the body-weight and age of a patient and the administration method but one skilled in the art can suitably select them. If said compound is encodable by a DNA, said DNA can be inserted into a vector for gene therapy and perform the therapy. The dosage and method of administration vary according to the body-weight, age, and symptoms of a patient but one skilled in the art can select them suitably.
For example, although there are some differences according to the symptoms, the dose of a compound that binds with the transcriptional regulatory factor of the present invention and regulates its activity is about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult (weight 60 kg).
When administering parenterally in the form of an injection to a normal adult (weight 60 kg), although there are some differences according to the patient, target organ, symptoms and method of administration, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per day and more preferably about 0.1 to about 10 mg per day. Also, in the case of other animals too, it is possible to administer an amount converted to 60 kg of body-weight.
This invention also features a DNA containing at least 15 nucleotides, which can specifically hybridize with DNA encoding the xe2x80x9cTCoA1xe2x80x9d protein. The term xe2x80x9cspecifically hybridizexe2x80x9d as used herein, indicates that cross-hybridization does not occur significantly with DNA encoding other proteins, in the above-mentioned hybridizing conditions, preferably under stringent hybridizing conditions. Such DNA includes, probes, primers, nucleotides and nucleotide derivatives (for example, antisense oligonucleotides and ribozymes), which specifically hybridize with DNA encoding the protein of the invention or its complementary DNA.
The present invention includes an antisense oligonucleotide that hybridizes with any site within the nucleotide sequence of SEQ ID NO:2 or 9. This antisense oligonucleotide is preferably that against the at least 15 continuous nucleotides in the nucleotide sequence of SEQ ID NO:2 or 9. The above-mentioned antisense oligonucleotide, which contains an initiation codon in the above-mentioned at least 15 continuous nucleotides, is even more preferred.
Derivatives or modified products of antisense oligonucleotides can be used as antisense oligonucleotides. Examples of such modified products are, lower alkyl phosphonate modifications such as methyl-phosphonate-type or ethyl-phosphonate-type, phosphothioate modifications and phosphoramidate modifications.
The term xe2x80x9cantisense oligonucleotidesxe2x80x9d as used herein means, not only those in which the entire nucleotides corresponding to those constituting a specified region of a DNA or mRNA are complementary, but also those having a mismatch of one or more nucleotides, as long as DNA or mRNA and an oligonucleotide can specifically hybridize with the nucleotide sequence of SEQ ID NO:9.
Such DNAs are indicated as those having, in the xe2x80x9cat least 15 continuous nucleotide sequence regionxe2x80x9d, a homology of at least 70% or higher, preferably at 80% or higher, more preferably 90% or higher, even more preferably 95% or higher. The algorithm stated herein can be used to determine homology. Such DNAs are useful as probes for the isolation or detection of DNA encoding the protein of the invention as stated in a later example or as a primer used for amplifications.
The antisense oligonucleotide derivative of the present invention, acts upon cells producing the protein of the invention by binding to the DNA or mRNA encoding the protein and inhibits its transcription or translation, promotes the degradation of the mRNA, inhibiting the expression of the protein of the invention resulting in the inhibition of the protein""s function.
The antisense oligonucleotide derivative of the present invention can be made into an external preparation such as a liniment and a poultice by mixing with a suitable base material, which is inactive against the derivatives.
Also, as needed, the derivatives can be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers, stabilizers, preservatives, pain-killers, and such. These can be prepared by following usual methods.
The antisense oligonucleotide derivative is given to the patient by, directly applying onto the ailing site or by injecting into a blood vessel so that it will reach the site of ailment. An antisense-mounting medium can also be used to increase durability and membrane-permeability. Examples are, liposome, poly-L-lysine, lipid, cholesterol, lipofectin or derivatives of these.
The dosage of the antisense oligonucleotide derivative of the present invention can be adjusted suitably according to the patient""s condition and used in desired amounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered.
The antisense oligonucleotide of the invention inhibits the expression of the protein of the invention and thereby useful for suppressing the biological activity of the protein of the invention. Also, expression-inhibitors comprising the antisense oligonucleotide of the invention are useful in the point that they can inhibit the biological activity of the protein of the invention.